Hybrid yeast cell lines for high level production of recombinant protein

ABSTRACT

This disclosure provides improved cell lines for manufacture of protein, considerably reducing the cost of commercial production. The cell lines are obtained by selecting cells from a mixed population for one or more characteristics that support protein production on a non-specific basis, such as the level of endoplasmic reticulum, Golgi apparatus, and/or other desired phenotypic features, compared with other cells in the starting mixture. Particularly effective producer cell lines can be obtained by preparing the cells for functional selection by making cell hybrids. A gene encoding a heterologous protein of interest may be transfected into the cells before or after one or more cycles of fusion and selection. Depending on the protein product being expressed, cell lines may be obtained that produce eight grams or more of protein per liter of culture fluid.

REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.16/440,776, filed Jun. 13, 2019 (pending), which is acontinuation-in-part of U.S. application Ser. No. 15/254,852, filed Sep.1, 2016 and issued as U.S. Pat. No. 10,329,594 on Jun. 25, 2019, whichclaims the priority benefit of U.S. provisional application 62/213,880,filed Sep. 3, 2015. U.S. application Ser. No. 16/440,776, is also acontinuation of international patent application PCT/US2019/036379,filed Jun. 10, 2019. The aforelisted priority applications are herebyincorporated herein by reference in their entireties for all purposes.

FIELD OF THE INVENTION

This application relates generally to the production of protein. It alsorelates to the modification and selection of cells and to transfectionof such cells with a gene of interest to obtain cell lines for proteinproduction at high productivity with improved biological andpharmacological characteristics.

BACKGROUND

Biological agents constitute a continually growing proportion of themarket for pharmaceuticals. They have higher specificity than otheragents, leading to more targeted efficacy with fewer side effects. Withit comes a burgeoning need for improved means of industrial production,with greater productivity and lower cost.

Some therapeutic proteins have a therapeutic dose and dosing schedulethat may require more than 10 grams of protein per patient per year.Current levels of protein production are generally no more than 4 g perliter of culture fluid, and are more typically less than 2 g per liter.To supply the market for a particular protein product, it may benecessary to produce 400,000 kg per year. This means that 100 millionliters of culture medium would need to be processed—about the volume of40 Olympic® sized swimming pools—which in turn would require severaldedicated $1 billion manufacturing facilities.

Recent advances in mammalian protein production are discussed in A. D.Bandaranayake and S. C. Almo, FEBS Lett 2014, 588(2): 253-260; and T.Lai et al., Pharmaceuticals 2013, 6:579-603. Cell engineering andcultivation of Chinese Hamster Ovary (CHO) cells is reviewed in T. Omasaet al., Current Pharmaceutical. Biotechnology, 2010:11, 233-240; C. A.Wilkens and Z. P. Gerdzen, PLOS ONE, Mar. 13, 2015; and J. Y. Kim etal., Appl. Microbiol. Biotechnol. 2012, 93:917-930. Multiplex genomeengineering using systems such as CRISPR/Cas 9 is reviewed by L. Cong etal., Science 2013, 339(6121):819-823; Y. Huang et al., J. Immunol.Methods 2007, 322:28-39; J. S. Lee et al., Science Reports, Feb. 25,2015; and P. Mali et al., Nat. Methods 2013, 10(10):957-963.

U.S. Pat. No. 5,607,845 (Spira et al., Pharmacia & Upjohn) proposed amethod for obtaining an increased production of a producing cell lineusing a fusion protocol. U.S. Pat. No. 6,420,140 (Hori et al., AbgenixInc.) proposed production of multimeric protein by a cell fusion method.Genome editing in CHO cells using CRISPR/Cas9 and CRISPy is reviewed byC. Ronda et al., Biotechnol. Bioeng. 2014, 111:1604-1616.

None of the technology described so far has the features and benefits ofthe technology of this invention, as described in the sections thatfollow.

SUMMARY OF THE INVENTION

Using previously available technology, production of therapeuticproteins (such as antibodies) has been expensive, requiring largevolumes of culture medium and complex infrastructure. This disclosureprovides substantially increased protein production yields on a per-cellbasis, reducing the cost of commercial production and potentiallyimproving product quality.

Model cell lines described in this disclosure are adapted for highlevels of protein production. In principle, the invention can beimplemented on any originating eukaryotic cell line, including but notlimited to mammalian cells, insect cells, and yeast cells. The cells arescreened for one or more characteristics that support protein productionon a basis that is not necessarily specific for a particular protein:for example, the density of endoplasmic reticulum in the cell, thedensity of Golgi apparatus, and/or the level of other desired phenotypicfeatures, compared with other cells in the starting mixture. Theselected cells have increased capacity to produce protein or other geneproduct from a transgene. The selected cells may or may not showincreased production from endogenous genes, since endogenous genes aresubject to further regulatory constraints. A gene encoding a therapeuticprotein is typically transfected into the cells before, after, or duringone or more cycles of selection. Depending on the chosen protein, celllines may be obtained that produce eight grams of protein per liter ormore of culture fluid.

One aspect of this invention is a method of obtaining a cell lineadapted for high-level production of protein-based pharmaceuticals. Theoriginating cell population is typically heterogeneous in terms ofprotein production capacity, and/or it may be treated in a manner suchthat at least some of the cells contained therein have an ability toproduce an increased amount of protein per cell than the cell populationas a whole. For example, a mixture of cells is treated such that themixture forms one or more cell hybrids, each comprising all or part ofthe genome of two or more cells from the mixture. Cells are selectedfrom the population to obtain a producer cell population that isenriched for a higher density of one or more subcellular organelles thatsupport increased production and/or secretion of protein, compared withother cells in the starting mixture. The originating mixture may consistessentially of cells from a single cell line, exemplified by ChineseHamster Ovary (CHO) cells, or a combination of two or more differentcell lines.

When this disclosure refers to a “producer cell line,” what is meant isa cell line that is suitable for production of a gene product, such asfor commercial use or sale. The technology put forth in this disclosureexplains how to obtain a producer cell line with special properties thatenable the cells to produce the gene product at a high level (per cell,or per culture volume) and/or with particular features of interest. Acell line that has been selected according to this technology may or maynot contain a recombinant transgene for production of a particularproduct, since the transgene can be introduced before or afterselection. Whether or not a transgene is present, a cell line created inaccordance with this invention has the special properties that enable itto be a high level producer of product once the transgene has beenintroduced into the cell. Such special properties may include a relativeenrichment for intracellular organelles involved in protein production,such as endoplasmic reticulum or Golgi apparatus. The capability of thecells for high level production from a transgene does not necessarilyimply that the cells also have a capability for high level productionfrom endogenous genes: in fact, if the increased production capabilityis selective for producing a target gene product encoded by thetransgene, the purity of the target (compared with total cellularproduction) will also be increased, which may facilitate purification.

The method for selecting suitable high producer cells may include one ormore of the following procedures in any combination:

-   -   selecting individual cells or hybrids that have a relatively        high density of endoplasmic reticulum per cell, compared with        other cells in the mixture;    -   selecting individual cells or hybrids that have a relatively        high density of Golgi apparatus, compared with other cells in        the mixture;    -   incubating cells with a vital dye that stains endoplasmic        reticulum and/or Golgi, and sorting cells according to the        amount of the vital dye associated with each cell;    -   expressing a fusion protein in cells in the mixture, wherein the        fusion protein contains a peptide that generates an optical        signal (such as GFP or luciferase) fused with a peptide that is        processed by the endoplasmic reticulum and/or the Golgi        apparatus, whereupon cells can be selected that express the        optical signal at a higher level than other cells in the        mixture.

The method for obtaining the high producer cells may further compriseone or more of the following procedures:

-   -   selecting cells that grow faster, or that grow better under        specified culture conditions;    -   binding the cells with antibody specific for a cell surface        ligand (the antibody optionally labeled or linked to a        particle), and selecting cells labeled with the antibody,        thereby obtaining a subpopulation that is enriched for cells        that express the ligand;    -   selecting cells that produce a relatively high level of a marker        protein, compared with other cells in the mixture, wherein the        marker protein is secreted from the cell and/or expressed on the        cell surface, such as secreted alkaline phosphatase or secreted        luciferase;    -   selecting cells that produce a preferred glycosylation pattern        or density on a marker protein, compared with other cells in the        mixture; and    -   culturing the producer cell population; and re-sorting cells        therein for the same feature, thereby obtaining a subpopulation        that is further enriched for cells in which an increased density        of the subcellular organelles is stably inheritable.

A producer cell line for a particular target protein can be obtainedaccording to this disclosure, for example, by transfecting cells from acell line that has already been selected for high levels of proteinproduction with a transgene gene encoding the target protein.Optionally, further selection for high levels of production can continueafter transfection. Alternatively, a transgene encoding a protein ofinterest can be transfected into a starting cell population, followingwhich cells are selected that produce a high level of protein productexpressed from the gene of interest, compared with other cells in themixture. In either case, the transgene may be inserted into the genomeof the cells either by random insertion, or at a location that ispre-selected as permitting or supporting a high level of transcription,compared with other locations in the genome. A producer cell line forthe target protein can then be established from the transfected andselected cells.

By way of example, the gene of interest may encode an antibody heavychain, an antibody light chain, or a single-chain antibody. The producercell line may express both an antibody heavy chain and an antibody lightchain that combine to produce an antibody having a desired specificity.The producer cell line may express a therapeutic enzyme, a hormone, agrowth factor, or a protein that is a naturally occurring component ofblood.

Another aspect of the invention is a cell line produced according to themethods provided in this disclosure that has been selected for highlevels of protein production—either before or after it has beengenetically modified to express a target protein. Such a cell line mayhave features selected from the following: a genome that contains partor all of the genome of two or more parental cell lines, a higherconcentration of endoplasmic reticulum and/or Golgi apparatus comparedwith any of the parental cell lines, and a capacity to produce aparticular level of protein from one or a combination of recombinantlyinserted genes, quantitated as described later in this disclosure. Theproducer cell line may or may not be clonal.

Aspects of the invention that are of current commercial interest to theinventor are indicated by the appended claims. Other aspects of theinvention will be apparent from the description that follows.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cell frequency profile for endoplasmic reticulum (ER)staining in native CHO cells, compared with CHO cell autotypic hybrids.

FIG. 2 shows the relative level of expression of alkaline phosphatasetransfected into native CHO cells, compared with CHO cell autotypichybrids. The expression in the fused cells shows over 4-fold improvement(p<0.05).

DETAILED DESCRIPTION

This disclosure provides improved cell lines for manufacture ofprotein-based pharmaceutical agents, considerably reducing the cost ofcommercial production. The cell lines are obtained by selecting cellsfrom a mixed population for one or more characteristics that supportprotein production on a non-specific basis, such as the level ofendoplasmic reticulum, Golgi apparatus, and/or other desired phenotypicfeatures, compared with other cells in the starting mixture.Particularly effective producer cell lines can be obtained by preparingthe cells for functional selection by making cell hybrids. A geneencoding a therapeutic protein of interest may be transfected into thecells before or after one or more cycles of fusion and selection.

Context

New biopharmaceutical products are coming on line at the rate of about100 per year, while competition in the production of biosimilarscontinues to increase. There is a clear need for technology that canreduce the culture volume and cost required for production of theseproducts.

This disclosure provides technology that allows product of proteinbiologicals at a productivity that surpasses current standards:potentially as much as 8 g per liter or more. The high efficiencyproducer cell lines described here can be used for industrialapplicability in several ways. Regarding biosimilars, companies withbrand-name products could maintain a marketing advantage by loweringtheir product cost structure. Similarly, companies producing biosimilarswould compete with the brand-name products on cost. The cell linesprovide considerable production flexibility by increasing capacity ofexisting plants; allowing production of more protein from fewer orsmaller facilities; and reducing cost and time-to-clinic for newproducts.

Producer cell lines that generate high levels of a target protein areobtained by screening and sorting a mixed cell population for individualcells that are better equipped for greater or more rapid production ofprotein on a per-cell basis.

Making Cell Hybrids

Individual high producer cells can be selected from any cell populationthat is heterogeneous in this respect, as described in the section thatfollows. Many single cell lines (such as CHO cells) are sufficientlydiverse at the outset in terms of gene content and intracellularapparatus in the proliferating cell population that they can be sortedand selected for high producer cells directly from a standard culture.

Optionally, to improve final product yield or enhance the sortingprocess, the user may prepare cells for sorting by taking one or acombination of techniques that will either enhance heterogeneity oflevels of protein production within the cell population, or generallyincrease the levels of protein production for the cells population as awhole, or a subpopulation thereof. Suitable techniques are those thatalter the genome of the cells, for example, to increase or shuffle genesthat contribute to the intracellular machinery involved in proteinproduction or processing. Altering or shuffling the genome in thismanner may yield many genetic variants with one or more of a variety ofdifferent properties, including levels of protein production and growthrate.

The maker of this invention has discovered that cells suitable forprotein production can attain a higher level of production by fusingwith other cells. Without limiting practice of the invention, it ishypothesized that fusing two cells together is partly additive in termsof the components, genetics, or genetic control of the cells thatparticipate in protein production. It is beneficial if the improvedcharacteristics breed true. Accordingly, after cells are fused, they aretypically subject to multiple rounds of culturing and selection forphenotypic characteristics of interest. The resulting cells may beaneuploid or otherwise retain all or part of the genomes of parentalcells that encode cell components involved in protein production.

Model cells suitable for fusion are cell lines that have already beenemployed for industrial protein production, such as CHO cells, mousemyeloma NS0 cells, mouse myeloma SP2/0 cells, Human Embryonic Kidney(HEK) 293 cells, and Baby Hamster Kidney (BHK-21) cells. Also suitableare other Chinese Hamster cell types (for example, breast and livercells that make secreted protein), human cell lines, and invertebratecells, such as insect and mollusk cells that may have desiredglycosylation properties. In the context of this disclosure, a “cellline” is a population of cells that can be propagated continually,extensively, or indefinitely in tissue culture. A starting cell line istypically heterogeneous in terms of one or more phenotypic features thatrelate to the amount of protein from a transgene that the cell willproduce. When cultured, a producer cell line obtained according to thisdisclosure may produce progeny that are heterogeneous, substantiallyhomogeneous, or clonal.

Cell fusion is performed by obtaining a cell mixture of cells to befused: (a plurality of cells from one cell line, or more than one cellline, or a mixture of at least one cell line and at least one primarycell population. The cell mixture is then subjected to an appropriatefusion protocol: for example, by culturing under culture conditions thatpromote the formation of hybrids, by conducting an electrofusion, bycombining with a fusogenic virus such as Sendai virus, by placing cellsinto contact (for example, by gentle centrifugation), by treating with afusogenic agent such as polyethylene glycol (PEG), or using anyeffective combination thereof.

For purposes of this disclosure, cells that have been made by fusing twoor more cells together may be referred to as autotypic hybrids (cellsfrom the same cell line fused together), isotypic hybrids (cells havingthe same genotype), allotypic hybrids (cells from different individualsof the same species having different genotypes), and xenotypic hybrids(cells from different species). Autotypic hybrids are typically formedusing a population of cells that consists essentially (that is, at least99%) of cells from a single cell line. The other types of hybrids aretypically formed using cell populations from two or more cell lineswhich have potentially complementary properties. The disclosure alsoincludes the fusion of one or more cell populations isolated or obtainedfrom primary sources with themselves or with established or cloned celllines.

Cells may be fused into hybrids using any suitable technique. Forexample, cells may be cultured in the presence of a fusogenic agentand/or under culture conditions that promote the formation of hybrids,or may be forced into contact, for example, by gentle centrifugation,optionally in combination with a fusogenic agent such as polyethyleneglycol (PEG). Typically a fused cell is obtained by fusing two cellstogether, although fusion of three or more cells is possible. It isrecognized that fusion of two different cell populations will result inmixed cell products (isotopic, allotypic, or xenotypic hybrids,depending on the parental cell lines), and autotypic hybrids. Autotypicor isotopic hybrids can be separated from allotypic or xenotypichybrids, if desired, using fluorescently labeled or surface boundantibody specific for a ligand expressed on one of the cell lines in themixture, but not another.

All such combinations come within the scope of this invention, unlessexplicitly indicated otherwise. It may be beneficial to repeat the cellfusion within a population of hybrids to enhance the effect further,and/or cross-hybridize with other cell lines to imbue the ultimate cellline with additional beneficial characteristics. Thus, the fusion andselection steps may be done iteratively twice, three or four times, ormore.

Selecting High Producer Cell Lines

A valuable insight of this disclosure is the idea that proteinproduction can be increased by selecting cells from a mixed cellpopulation for higher levels subcellular machinery or biochemistry thatsupport increased protein production, compared with other hybrids orparental cells in the starting mixture. At least one of the phenotypicfeatures is selected that is not necessarily specific for production ofa particular protein. The feature is not simply the level of expressionof a protein of interest or a surrogate. Rather, it is a feature thatsupports production of a wide range of different proteins. Such featuresinclude the relative density of subcellular organelles, particularlythose involved in secretion of protein from the cell, and the relativelevel or concentration of enzymes that help finish or secrete a varietyof different proteins.

Subcellular organelles involved in production of protein include theendoplasmic reticulum (ER) and the Golgi apparatus. Either or both ofthese can be measured and used as a basis for sorting or selectionwithout damaging the cell using a vital dye, and the cells can beselected on the basis of the amount of dye that is associated.

Such dyes can be obtained commercially, for example from the companyMolecular Probes. Examples of vital dyes for ER include:

ER-Tracker™ Blue-White DPX (E12353)

ER Tracker™ Green (glibenclamide BODIPY® FL) (E34251)

ER-Tracker™ Red (glibenclamide BODIPY® TR) (34250)

DiOC₆ (D273)

DiOC₅ (D272).

Vital dies for Golgi apparatus include:

NBD C6-6-ceramide (N1154)

NBD C6-sphingomyelin

BODIPY® FL C5-cerimide (D3521)

BODIPY® TR ceramide (D7540)

Alternatively or in addition, the user can test expression-basedlabeling systems that would introduce a fluorescent protein targeted toER or Golgi. They are fusion proteins comprising a portion thatexpresses an optical label, fused with a protein sequence that targetsor is processed by the organelle to be labeled. Examples include thefollowing:

Invitrogen:

CellLight™ ER-GFP (C10590)

CellLight™ ER-GFP (C10591)

CellLight™ Golgi-GFP (C10592)

CellLight™ Golgi-GFP (C10593)

Evrogen:

pmKate2-ER (FP324)

pFusionRed-ER (FP420)

pTagRFP-Golgi (FP367)

pTagRFP-Golgi (FP367)

pFusionRed-Golgi (FP419)

Clontech:

pDsRed2-ER Vector (632409)

pDsRed-Monomer-Golgi Vector (632480)

pAcGFP1-Golgi Vector (632464)

After staining with any of these dyes, cells may be selected (forexample, by flow cytometry and sorting) that have on average a level ofstaining that is at least 1.2, 1.5, 2, or more than 2-fold higher thanthe parental cell line or lines, in terms of staining, for example, forER, Golgi, or an optically labeled gene product.

Other features to select for may include but are not limited tophenotypic features, immunological features, and levels of proteinproduction. Immunological features may include expression of a desiredligand by the cell (for example, secreted by the cell or expressed onthe surface). Cells having an average level of expression of suchmarkers that is at least 1.5, 2, 3, or 5-fold higher than the parentalline can be selected, for example, by direct or indirect antibodylabeling followed by FACS, or by binding to and releasing fromantibody-coated microbeads. Immunological markers of interest includeligands that participate in production of secreted protein, such asglycosylation enzymes. Other sorting methods that can be used to screencells according to this disclosure may include PCR-activated cellsorting, fluorescence in situ hybridization flow cytometry (FISH-PC), orFISH followed by laser capture.

Simultaneously or as a separate step, individual cells can also beselected from a mixed cell population for features that are desired formanufacturing purposes: such as cells that grow better under specifiedculture conditions, or that express relatively lower levels of one ormore undesired contaminants.

To generate a cell line that is sufficiently stable to be used formanufacturing biological agents, the selected cells can be grown inculture through several cell divisions, and then re-tested to see if thedesired feature is stable, for a total of two, three, or more than threetimes for each desired feature.

Transfecting Cells with a Gene of Interest

To generate a cell line expressing a protein of interest (a targetprotein), producer cells or their precursors can be transfected with agene encoding the protein under control of a ubiquitous or mammalianpromoter that causes expression in the host cell line. The level ofproduction of the target protein can be determined in the course ofprocessing using a transient transfection method to insert a proteinexpression cassette. Alternatively or subsequently, permanenttransfection can be done that integrates the gene of interest or amarker gene into the genome of the cell line. Transfection can be doneusing liposome-based reagents (for example, Lipofectamine™ 2000 orFuGENE™ 6), calcium phosphate, electroporation, or infection with anadenovirus, retrovirus or lentivirus based vector.

Following transfection, the cells are tested for production or secretionof the target protein (typically after cloning or limiting dilutionculture): for example, by enzyme-linked immunosorbent assay (ELISA).Cells or clones having increased production of the desired protein areselected. The objective can be an increase in protein production that is1.5, 2, 4, 8, 12, 16, or 20-fold higher than the parental cell line;and/or production at a level of 6 g, 8 g, 10 g, 12 g, 15 g, or 20 g perliter of culture fluid under typical manufacturing conditions. Theprotein of interest can also be tested for other desiredcharacteristics, such as the quality of sialylation or other aspects ofglycosylation.

In principle, the transfection can be done either before, during, orafter one or more cycles of fusion and selection for other features. Forexample, the fusion and selection can be done before transfection withthe gene of interest, thereby establishing a parental cell line suitablefor high-level production of a protein of the user's choice.Alternatively, the transfection can be done into the originatingparental cell line, and used to track production levels duringsubsequent fusion and sorting steps, or to provide another basis forsuch sorting. Alternatively, the transfection can be done as anintermediate step, wherein the cells have already been subject to one ormore cycles of fusion and selection for some other feature such as ER orGolgi, the resulting hybrid is transfected to express a protein ofinterest, and then subjected to further cycles of fusion and selectionfor expression of the protein of interest and/or other features referredto earlier in this disclosure.

The protein of interest can be the biological agent that is intended formanufacture: for example, an antibody heavy chain, an antibody lightchain, a single-chain antibody, a therapeutic enzyme, a hormone, agrowth factor, or a protein that is normally a blood component.

Another option is to develop a cell line using a marker protein as aproxy for the protein that ultimately will be manufactured: for example,secreted alkaline phosphatase or secreted luciferase. Again, thetransfection can be done before, during, or after multiple cycles offusion and selection, optionally using the level of expression of themarker as the selection criteria in one or more of the cycles. Thiscreates a parental cell line that is optimized for expression of themarker protein, with the expectation that the beneficial characteristicsof the cell line will be retained after further genetic alteration toproduce a biological product of commercial interest.

Ultimately, once a cell line has been developed having a desired levelof expression of the marker protein, the marker is then replaced withthe protein of interest. Transfection can again be done randomly intothe genome, using the techniques listed above, and expression of themarker protein is curtailed. Alternatively, the gene for the markerprotein can be substituted with a gene that encodes the protein ofinterest using a targeted integration technique. Such techniquescomprise, for example, CRISPR/Cas9, a zinc-finger recombinase (ZFR), ora transcription activator-like effector nuclease (TALEN). That way, thegene of interest is inserted into the genome of the cells from theproducer cell line or the mixture at a location that is pre-selected aspermitting or supporting a high level of transcription, compared withother locations in the genome.

For more information on the use of targeted integration techniques, thereader may refer to L. Cong et al., Science 2013, 339(6121):819-823; Y.Huang et al., J. Immunol. Methods 2007, 322:28-39; J. S. Lee et al.,Science Reports, Feb. 25, 2015; and P. Mali et al., Nat. Methods 2013,10(10):957-963; and C. Ronda et al., Biotechnol. Bioeng. 2014,111:1604-1616.

Incorporation of Additional Features

The system and techniques provided in this disclosure can be combinedwith one or more alternative strategies to enhance cell growth orprotein production for purposes of manufacture. Such techniques includevector and expression platform engineering, omics-based approaches,advances in gene delivery and integration, enhancement of proteinproduction using chromatin opening elements, improvements in clonescreening strategy, and so on.

Such techniques are discussed, for example, in A. D. Bandaranayake andS. C. Almo, FEBS Lett 2014, 588(2): 253-260; T. Lai et al.,Pharmaceuticals 2013, 6:579-603; T. Omasa et al., CurrentPharmaceutical. Biotechnology, 2010:11, 233-240; C. A. Wilkens and Z. P.Gerdzen, PLOS ONE, Mar. 13, 2015; J. Y. Kim et al., Appl. Microbiol.Biotechnol. 2012, 93:917-930; and C. Ronda et al., Biotechnol. Bioeng.2014, 111:1604-1616.

One such feature suitable for incorporation is rapid proliferation.Mixed cell populations can be screened at any time during development ofthe producer cell line, either concurrently or as a separate step fromthe selection of cells that are equipped for high levels of proteinproduction on a per-cell basis, based in content of endoplasmicreticulum and/or Golgi. Non-viable or slow-growing cells are removed ordiluted out from the population during selection for faster growth.

By way of illustration, cells are cultivated in an appropriate culturemedium and under appropriate conditions. A typical seed concentration ofcells would be 2×10⁵ cells/mL. The cells are cultivated for two days,then sub-cultivated by diluting cells to a concentration of 2×10⁵cells/mL. Repeat as desired, so that slower-growing cells arediluted-out. As the proportion of faster-growing cells in the mixedculture increases, the time between sub-cultivation steps can bedecreased and/or the extent of cell dilution at each step can beincreased.

Characteristics of Producer Cells

A cell line or mixed cell population that has been selected for highlevels of protein production may be characterized in comparison with theparental or originating cell line by any one or more of severaldifferent parameters. For example, the selected cells may have: (1) agenome that is more aneuploid than the starting cells, containing partor all of the genome of two or more parental cell lines (which may ormay not be the same), (2) a higher concentration of endoplasmicreticulum and/or Golgi apparatus compared with any one or all of theparental cell lines (for example, between 2 to 5-fold or 4 to 8 fold, ormore than 2-, 4-, or 8-fold higher), (3) a capacity to produce a levelof target protein per cell or per liter of culture fluid that issubstantially higher than the parental cell line (for example, between 2to 5-fold or 4 to 8 fold, or more than 2-, 4-, or 8-fold higher), (4) acapacity to produce a particular amount of target protein per cell (forexample, more than 50, 65, 75, 100, 150, 200, 300, or 500 pg/cell/day,or from 50 to 200 or 75 to 300 pg/cell/day); or (5) a capacity toproduce a certain amount of target protein per volume of culture fluid(for example, at least 5, 8, 12, 20, or 30 grams, or between 8 and 20 orbetween 10 and 50 grams of protein per liter of culture fluid.

For the purpose of making such comparisons, the producer cell line canbe compared with a standardized population of the original cell line,either kept on hand, as part of the same system, or obtained from areference source. For example, CHO derived producer cells may becompared with CRL-12023 cells from the American Type Culture Collection(ATCC®). This disclosure includes systems for high-level production ofprotein-based pharmaceuticals, comprising both a starting cell line, anda producer cell line derived therefrom that has a relatively highdensity of endoplasmic reticulum and/or Golgi apparatus per cell, asdetermined, for example, using one or more of the vital dyes listedabove.

Benefits of this Technology

Depending on the mode of practice and application, aspects of theinvention described in this disclosure can provide any of the followingbenefits in any combination:

-   -   reduce the need to enlarge or build new GMP production        facilities as market size increases;    -   provide GMP production of kilogram quantities of finished        protein product with relatively small or fewer bioreactors,    -   reduce the cost of production of proven biological agents;    -   create production cell lines suitable for high-level expression        of a family of desired biological agents;    -   decrease cloning or selection steps that are needed following        integration of the gene to be expressed;    -   improve product quality (for example, glycosylation); and    -   provide high quality low volume research materials, reducing the        time to clinical trials.

EXAMPLES Example 1

The technology of the invention can be practiced using the K1 line ofCHO cells (ATCC® CCL-61). A population of CHO cells grown in culture isfused so as to make isotypic hybrids according to the followingprotocol:

-   -   1. Centrifuge 10⁷ cells.    -   2. Discard supernatant    -   3. Break the pellet by gently tapping the bottom of the tube    -   4. Add 100 μL of 50% PEG over the period of one minute, while        mixing the cells with a pipette tip    -   5. Continue stirring the cells for one additional minute    -   6. Add 100 μL of growth medium over one minute while mixing    -   7. Add 300 μL of growth over three minutes while mixing    -   8. Slowly add mL. of growth medium    -   9. Incubate at 37 degrees C. for five minutes    -   10. Centrifuge    -   11. Re-suspend the pellet in 20 mL of growth medium and transfer        to a 125-mL culture flask    -   12. Culture normally.

Alternatively, an electrofusion procedure is employed using ECM2001pulse generator (BTX). 10⁷ cells are centrifuged and resuspended in 1 mLof Cytofusion™ Medium C, then transferred into the fusion chamber. Cellsare aligned with an alternating current pulse of 150 V/cm for 10seconds. Cell fusion is triggered by a single square wave direct currentpulse of 1200 V/cm for 25 μsec. Cells are allowed to rest for 5 min.,centrifuged, then resuspended in growth medium and cultured normally.

Alternatively, a virus-induced fusion protocol may be employed. Variousprotocols exist using Sendai virus: for example, using a GenomONE™ HVJ-EKit (Cosmo Bio USA): Cells are centrifuged and resuspended in ice coldcell fusion buffer at 2×10⁵ cells/25 μL. 2.5 μL of an ice-cold HVJ-E(Sendai virus membranes) suspension is added to the cells and mixed bytapping. Mixture is incubated on ice for 5 min; then at 37 deg C. for 15min. Growth medium is added to the mixture and it is transferred into asix-well plate for culture.

Labeling and sorting for subcellular organelles can be done as follows.The cells are centrifuged and washed once with HBSS buffer. A 1 μMsolution of ER-tracker Green and/or ER-tracker Blue/White is prepared inHBSS. The cells are re-suspended in staining solution and incubated at37 deg C. for 30 minutes. The cells are then washed with PBS.

If cells are to be used for analytical FACS, they are re-suspended inPBS; if they are to be sorted, they are re-suspended in PBS supplementedwith 1% FBS. Ten percent of the viable population exhibiting the highestamount of staining with ER-Tracker dye was collected. The cells arecollected into tubes containing growth medium, centrifuged, re-suspendedin fresh medium, and then cultured normally.

Example 2

CHO-K1 cells were exposed to a PEG-assisted fusion procedure. The cellswere allowed to recover for one week, then the procedure was repeatedfor a total of three times. Following recovery from the third fusion,the cells were stained with vital ER-tracking dye (ER-Tracker™ Green(glibenclamide BODIPY® FL); Invitrogen, E34251) and sorted using aFACSAriaII™ cell sorter (BD Biosciences). Ten percent of the viablepopulation exhibiting the highest amount of staining with ER-Tracker dyewas collected. Following a two-week recovery in culture, the cells wereexposed to a final fusion, stained with ER-tracking dye, and analyzedusing a LSRII™ flow cytometer (BD Biosciences).

To measure protein production in the fused cells, and the parental CHOpopulation, the cells were transfected to express secreted alkalinephosphatase (SEAP). The transfection was performed as follows:

-   -   1. Centrifuge 10⁶ cells.    -   2. Discard supernatant    -   3. Resuspend in 100 μL Cell Line Nucleofector™ Solution T    -   4. Add 2 μg SEAP expression plasmid    -   5. Transfer to electroporation cuvette    -   6. Electroporate using Amaxa™ Nucleofector II and preset program        U-023    -   7. Add 0.5 ml growth medium    -   8. Transfer cells into 6-well plate containing mL. growth medium        per well

FIG. 1 is the FACS (florescence-activated cell sorting) profile of theCHO cells after fusion and staining for levels of endoplasmic reticulum(ER). Fused cells showed a higher average level of ER compared with thestarting CHO cell line.

FIG. 2 shows the transfection results (specific productivity of secretedalkaline phosphatase). The expression of the marker protein in the fusedcells is shows over 4-fold improvement.

For all purposes in the United States of America, each and everypublication and patent document referred to in this disclosure isincorporated herein by reference in its entirety for all purposes to thesame extent as if each such publication or document was specifically andindividually indicated to be incorporated herein by reference.

While the invention has been described with reference to the specificembodiments, changes can be made and equivalents can be substituted toadapt the invention to a particular context or intended use as a matterof routine experimentation, thereby achieving benefits of the inventionwithout departing from the scope of what is claimed.

The invention claimed is:
 1. A method of obtaining a hybrid yeast cellline adapted for high-level production of protein, comprising: (a)culturing a starting mixture of yeast cells under conditions whereby oneor more cell hybrids are formed, each comprising two or more cells fromthe mixture; then (b) sorting cells in the mixture according to densityof endoplasmic reticulum and/or Golgi apparatus per cell; and (c)selecting and recovering cell hybrids from the mixture that have arelatively high density of endoplasmic reticulum and/or Golgi apparatusper cell; thereby obtaining a yeast cell line that supports increasedproduction and/or secretion of protein compared with parental cells inthe starting mixture.
 2. The method of claim 1, wherein the mixturecultured in step (a) consists essentially of cells from a single yeastcell line.
 3. The method of claim 1, wherein the mixture cultured instep (a) comprises two or more different yeast cell lines.
 4. The methodof claim 1, wherein step (c) includes selecting and recovering cellhybrids that have a relatively high density of endoplasmic reticulum percell, compared with other hybrids or parental cells in the mixture. 5.The method of claim 1, wherein step (c) includes selecting andrecovering cell hybrids that have a relatively high density of Golgiapparatus, compared with other hybrids or parental cells in the mixture.6. The method of claim 1, wherein step (b) includes incubating cellswith a vital dye that stains endoplasmic reticulum and/or Golgi, andsorting cell hybrids according to the amount of the vital dye associatedwith each hybrid.
 7. A method of obtaining a hybrid yeast cell lineadapted for high-level production of protein, comprising: (a) culturinga mixture of yeast cells under conditions whereby the mixture forms oneor more cell hybrids, each comprising two or more cells from themixture; then (b) expressing a fusion protein in cells in the mixture,wherein the fusion protein contains a fluorescent or bioluminescentpeptide that generates an optical signal fused with a peptide that isprocessed by endoplasmic reticulum and/or Golgi apparatus; and (c)selecting and recovering cells that express the optical signal at ahigher level relative to other cells in the mixture; thereby obtaining ayeast cell line that supports increased production and/or secretion ofprotein compared with other hybrids or parental cells in the startingmixture.
 8. The method of claim 1, comprising selecting cell hybridsthat produce a relatively high level of a marker protein, compared withother cell hybrids in the mixture, wherein the marker protein issecreted from the cell and/or expressed on the cell surface.
 9. Themethod of claim 1, further comprising culturing the yeast cellpopulation, and re sorting cell hybrids therein according to the densityof endoplasmic reticulum and/or Golgi apparatus in the cell hybrids,thereby obtaining a subpopulation that is enriched for cell hybrids inwhich an increased density of the subcellular organelles is stablyinheritable.
 10. The method of claim 1, wherein cells are furtherselected for increased growth rate compared with the parental cells inthe starting mixture.
 11. The method of claim 1, further comprisingtransfecting the cells with a gene of interest to obtain a transfectedcell population, and selecting transfected cells from the transfectedcell population that produce high levels of a protein product of thegene of interest, relative to other cells in the transfected cellpopulation.
 12. The method of claim 11, wherein the transfecting isperformed before step (b) or after step (c).
 13. A method of producing aprotein, comprising: obtaining a transfected cell population accordingto the method of claim 11, wherein the recombinantly inserted gene(s)encode part or all of the protein, and culturing the cells underconditions whereby the protein or a portion thereof is expressed by thegene transfected into the cells.
 14. The method of claim 13, wherein theyeast cell line expresses at least eight grams of protein per liter ofculture fluid from one or a combination of recombinantly inserted genes.15. A hybrid yeast cell line that has a relatively high density ofendoplasmic reticulum and/or Golgi apparatus per cell, which is therebyadapted for high-level production of a protein, wherein cells of theyeast cell line include a transgene encoding the intended protein thatis integrated into the genome of the cells; wherein the yeast cell linehas been prepared by the following process: (a) culturing a startingmixture of yeast cells under conditions whereby one or more cell hybridsare formed, each comprising two or more cells from the mixture; then (b)sorting cells in the mixture according to the density of endoplasmicreticulum and/or Golgi apparatus per cell; and (c) selecting andrecovering cell hybrids from the mixture that have a relatively highdensity of endoplasmic reticulum and/or Golgi apparatus per cell;thereby obtaining a yeast cell line that supports increased productionand/or secretion of protein compared with parental cells in the startingmixture.
 16. The yeast cell line of claim 15, wherein the yeast cellline expresses at least eight grams of protein per liter of culturefluid from the transgene.
 17. A hybrid yeast cell line, wherein eachcell of the cell line comprises: part or all of the genome of one ormore parental yeast cell lines; a higher concentration of endoplasmicreticulum and/or Golgi apparatus relative to any of the parental celllines; and a recombinantly inserted transgene; wherein the hybrid cellline has a capacity of producing a higher mass of protein per liter thanany of the parental cell lines.
 18. The hybrid yeast cell line of claim17, having the capacity of producing from the transgene at least eightgrams of protein per liter of culture fluid.
 19. A method of producing aprotein, comprising: obtaining cells from a hybrid yeast cell lineaccording to claim 17, wherein the transgene encodes part or all of theprotein, and culturing the cells under conditions whereby the protein ora portion thereof is expressed by the cells from the transgene.
 20. Themethod of claim 19, further comprising compounding the protein into apharmaceutical product for treatment of a human subject in need thereof.